New Phenolic Dimers from Plant Paeonia suffruticosa and Their Cytotoxicity and NO Production Inhibition

The Paeonia suffruticosa, known as ‘Feng Dan’, has been used for thousands of years in traditional Chinese medicine. In our chemical investigation on the root bark of the plant, five new phenolic dimers, namely, paeobenzofuranones A–E (1–5), were characterized. Their structures were determined using spectroscopic analysis including 1D and 2D NMR, HRESIMS, UV, and IR, as well as ECD calculations. Compounds 2, 4, and 5 showed cytotoxicity against three human cancer cell lines, with IC50 values ranging from 6.7 to 25.1 μM. Compounds 1 and 2 showed certain inhibitory activity on NO production. To the best of our knowledge, the benzofuranone dimers and their cytotoxicity of P. suffruticosa are reported for the first time in this paper.


Introduction
Paeonia suffruticosa is a perennial deciduous shrub belonging to the family Paeoniaceae. The dried root bark of P. suffruticosa is called 'Feng Dan' or 'Mudanpi' in China, which is used as a traditional Chinese medicine to clear pathogenic heat from the blood and promote blood circulation to remove blood stasis, as recorded in the Chinese Pharmacopoeia (2020 edition). Currently, pharmacological studies on P. suffruticosa demonstrate anti-inflammatory, antioxidant, and anti-tumor activities, as well as central nervous system and cardiovascular system protective activities [1][2][3]. In order to understand the bioactive metabolites, P. suffruticosa was chemically investigated, and more than 190 constituents were reported in the past ten years including phenolics, monoterpenes and glycosides, flavonoids, triterpenes, sesquiterpenes, and lignans. A pharmaceutical investigation on these metabolites demonstrated promising anti-inflammatory and anti-tumor properties; the representatives are paeonisides A and B, mudanpiosides C and F, and suffruticosol A [4][5][6][7][8].
In order to further clarify the chemical constituents and biological activities of genuine medicinal materials of 'Feng Dan' from Tongling (China), as well as part of our ongoing work on bioactive natural products from natural sources [9][10][11][12][13][14], the study on the chemical constituents of the root bark of P. suffruticosa was carried out. As a result, five new phenolics were isolated, namely, paeobenzofuranones A-E (1)(2)(3)(4)(5). Their structures ( Figure 1) were determined using extensive spectroscopic methods. All compounds were evaluated for their cytotoxicity against three human cancer cell lines including breast cancer MDA-MB-231, human myeloid leukemia HL-60, and colon cancer SW480. In addition, their anti-inflammatory activity by inhibiting NO production was also evaluated. Herein, the isolation, structural elucidation, and bioactivities of the compounds from P. suffruticosa are reported. evaluated. Herein, the isolation, structural elucidation, and bioactivities of the compounds from P. suffruticosa are reported.
Compound 4 was obtained as a white powder. The IR spectrum indicated that compound 4 possessed hydroxyl (3383 cm −1 ) and lactone (1708 cm −1 ) groups. Its molecular formula was determined as C17H16O4 by the HR-ESI-MS data analysis (m/z 285.11215 ([M + H] + , calcd. 285.11268). The 1 H and 13 C spectra data of compound 4 (Tables 1 and 2, Supplementary data) are partially the same as those of compound 1, except for the benzoyl and hydroxymethyl groups in compound 4. The interpretation of the 1 H and 13 C spectroscopic data of compound 4 implied one benzofuran part and one benzoyl. The locations of the benzofuran parts were assigned by the correlations revealed in the HMBC experiment ( Figure 2) between the 11-CH3 (δH 2.14) and C-5 (δC 148.9), C-7 (δC 110.6), and C-8 (δC 153.4); as well as from H-4 (δH 6.74) to C-5, C-8, and C-9 (δC 125.6); from H-3 (      (Tables 1 and 2), except for the presence of an additional methoxy at C-2 in compound 5, which was confirmed by the key HMBC correlation of H-12 (δH 3.48) with C-2 (δ C 111.2). A comprehensive analysis of the 2D NMR data indicated that other parts of compound 5 were the same as those of compound 4. The absolute configurations of C-2 and C-3 were established as 2S and 3S by the ECD calculations ( Figure 4). Thus, the structure of compound 5 was established as paeobenzofuranone E.

Bioactivity Analysis
Five new compounds were tested for their inhibitory activities on nitric oxide production in the model of lipopolysaccharide-activated macrophages. As shown in Table 3, compounds 1 and 2 showed comparable inhibitory activity with the positive control at the concentration of 50 µM. In addition, all compounds were evaluated for their cytotoxicity against the HL-60, SW480, and MDA-MB-231 cell lines. As shown in Table 4, compounds 2, 4, and 5 demonstrated cytotoxicity against three human cancer cell lines. In particular, they exhibited potent cytotoxicity against HL-60 cells, with IC 50 values of 6.8, 19.1, and 11.1 µM, compared to those of the positive control. In addition, compounds 4 and 5 showed no cytotoxicity to MDA-MB-231, indicating selectivity to the cancer cell lines. Table 3. Inhibitory activities of compounds 1-5 on NO production at 50 µM.

General Experimental Procedures
The UV spectra were obtained on a UH5300 UV-VIS Double Beam Spectrophotometer. The IR spectra were accessed using a Shimadzu Fourier transform infrared spectrometer with KBr pellets. The HRESIMS were measured on a Thermo Scientific Q Exactive Orbitrap mass spectrometer system. The NMR spectra ( 1 H, 13 C, and 2D NMR) were run on a Bruker Avance III NMR instrument at 600 MHz for 1 H and 150 MHz for 13 C NMR, while tetramethylsilane (TMS) was used as an internal standard. Column chromatography (CC) was executed on silica gel (200−300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Stockholm, Sweden), and reverse phase silica gel (20−45 µm, Fuji Silysia Chemical Ltd., Kasugai, Japan). Medium pressure liquid chromatography (MPLC) was applied to Biotage SP2 equipment, and the columns were packed with reverse phase silica gel (C 18 ). An Agilent 1260 series high-performance liquid chromatography (HPLC) system was used for the sample analysis (ZORBAX-SB C 18 column, 5 µm, 4.6 × 250 mm, flowing rate = 1 mL/min) and preparation (ZORBAX-SB C 18 column, 5 µm, 9.4 × 150 mm, flowing rate = 6 mL/min). All fractions were monitored using thin-layer chromatography (TLC) over GF 254 and silica gel 60 plates. The spots were visualized by using heating silica gel plates soaked with vanillin-sulfuric acid color component solvent.

Plant Material
The root barks of P. suffruticosa were collected in August 2021 from Tongling County, Anhui Province, People's Republic of China. It was identified by Zhenghui Li. (Associate Professor of South-Central Minzu University, Wuhan, China). A voucher specimen (2021123 FD) was deposited at the School of Pharmaceutical Sciences, South-Central Minzu University.

Cytotoxicity Assay
The cytotoxicity for the isolates was evaluated using the MTS assay. Briefly, 1 × 10 5 cells/mL from three human cancer cell lines, breast cancer MDA-MB-231, human myeloid leukemia HL-60, and colon cancer SW480, were seeded in 96-well plates. After 24 h incubation, the cells were treated with test compounds or cisplatin (DDP, positive control) at given concentrations (40, 8, 1.6, 0.32, 0.064 µM) for 48 h. The MTS was then added to each well, and the plates were stored for 4 h. The absorbance was read at 490 nm. The IC 50 (50% concentration of inhibition) was calculated using the Reed-Muench method [16,17].

NO Inhibitory Activity Assays
The mouse mononuclear macrophages RAW264.7 were seeded into 96-well plates, induced, and stimulated with 1 µg/mL LPS; at the same time, five new compounds with different concentrations to be tested were added. The drug-free group and the L-NMMA positive drug group were set approximately equal as a comparison. After the cells were cultured overnight, the medium was taken to detect the production of NO, and the absorbance was measured at 570 nm. The MTS was added to the remaining medium for cell viability assays to exclude the toxic effects of the compounds on the cells. The assays were performed as triplicate batch experiments. The NO production inhibition rate (%) = (OD 570nm of non-drug treatment group−OD 570nm of sample group)/OD 570nm of non-drug treatment group × 100% [18,19].

ECD Calculations
The conformers of the five calculated compounds were generated via MMFF in Chem-Draw. The ECD were calculated at the B3LYP/6-31+G(d,p) level in methanol with the PCM model. The calculated ECD curves and weighted ECD were all generated using SpecDis 1.71 based on the Boltzmann distribution theory, and the simulated spectra of all the predominant conformers were averaged to obtain the final conformationally averaged data [20]. All of the density functional theory (DFT) calculations were implemented using the Gaussian 16 software package with the Gaussian 09 default keyword. For the computational details of compounds 1-5, see the Supplementary Materials.

Conclusions
In the present study, the chemical investigation on Paeonia suffruticosa results in the isolation of five new benzofuran compounds, containing rare dimers (compounds 1-3) and hetero-dimers (compounds 4 and 5). Their structures were determined using extensive spectroscopic methods. This work represents the first report of new benzofuran dimers of P. suffruticosa and their cytotoxicity and broadens the horizon of the structural diversity of P. suffruticosa.
Author Contributions: T.F. and J.L. designed and guided the experiment; Q.M. performed the isolation and identification of the compounds and wrote the manuscript; S.T. and Y.Z. contributed to the isolation of these compounds; X.P. reviewed the manuscript; Z.L. obtained the plant material and identification; T.F. and J.L. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.